Correlation between microdilution, Etest, and disk diffusion methods for antifungal susceptibility testing of fluconazole against Candida sp. blood isolates

INTRODUCTION: Antifungal susceptibility testing assists in finding the appropriate treatment for fungal infections, which are increasingly common. However, such testing is not very widespread. There are several existing methods, and the correlation between such methods was evaluated in this study. METHODS: The susceptibility to fluconazole of 35 strains of Candida sp. isolated from blood cultures was evaluated by the following methods: microdilution, Etest, and disk diffusion. RESULTS: The correlation between the methods was around 90%. CONCLUSIONS: The disk diffusion test exhibited a good correlation and can be used in laboratory routines to detect strains of Candida sp. that are resistant to fluconazole.

Polymerase chain reaction and nested-PCR approaches for detecting Cryptosporidium in water catchments of water treatment plants in Curitiba, State of Paraná, Brazil

Introduction Cryptosporidium is an important protozoan cause of waterborne disease worldwide of concern to public health authorities. To prevent outbreaks of cryptosporidiosis, the monitoring of this parasite in drinking water is necessary. In the present work, the polymerase chain reaction (PCR) and nested-PCR techniques were used to detect Cryptosporidium in raw water from catchment points of four water treatment plants (WTP) in Curitiba, Paraná, Brazil. Methods First, DNA extraction techniques were tested in samples containing decreasing amount of oocysts in reagent water, and PCR and nested-PCR with specific primers for 18SSU rDNA of Cryptosporidium were conducted to determine their sensitivity. In reagent water, a commercial extraction kit provided the best analytical sensitivity, and PCR and nested-PCR allowed the detection of five and two oocysts, respectively, with the primers XIAOR/XIAOF and XIAO1F/XIAO2R. Results In the spiking experiments, only the PCR with the primers AWA995F/AWA1206R was successful at detecting concentrations of 0.1 oocysts/mL. Two catchments samples of raw water and/or water sludge from four WTPs were contaminated with Cryptosporidium. Conclusions The application of the techniques to monitor Cryptosporidium in water and detect contamination in water catchments of WTPs in Curitiba are discussed in the present work.

Investigation of class 1 integrons in Klebsiella pneumoniae clinical and microbiota isolates belonging to different phylogenetic groups in Recife, State of Pernambuco

Introduction The high prevalence of Klebsiella pneumoniae infections is related to the ability of K. pneumoniae to acquire and disseminate exogenous genes associated with mobile elements, such as R plasmids, transposons and integrons. This study investigated the presence of class 1 integrons in clinical and microbiota isolates of K. pneumoniae belonging to different phylogenetic groups and correlated these results with the antimicrobial resistance profiles of the studied isolates. Methods Of the 51 isolates of K. pneumoniae selected for this study, 29 were from multidrug-resistant clinical isolates, and 22 were from children's microbiota. The susceptibility profile was determined using the disk diffusion method, and class 1 integrons were detected through polymerase chain reaction (PCR). Results The results showed that none of the 22 microbiota isolates carried class 1 integrons. Among the 29 clinical isolates, 19 (65.5%) contained class 1 integrons, and resistance to sulfamethoxazole/trimethoprim was identified in 18 of these isolates (94.7%). Among the K. pneumoniae isolates with class 1 integrons, 47% belonged to the KpI phylogenetic group, and one isolate (14.3%) carrying these genetic elements belonged to the KpIII group. Conclusions The wide variety of detected class 1 integrons supports the presence of high rates of antimicrobial resistance, genetic variability, and rapid dissemination of beta-lactamase genes among K. pneumoniae clinical isolates in recent years in hospitals in Recife-PE, Brazil. The findings of this study indicate that the surveillance of K. pneumoniae integrons in clinical isolates could be useful for monitoring the spread of antibiotic resistance genes in the hospital environment.

The thermal effects on the methanol-to-olefins reaction: A modelling and experimental approach

With the projection of an increasing world population, hand-in-hand with a journey towards a bigger number of developed countries, further demand on basic chemical building blocks, as ethylene and propylene, has to be properly addressed in the next decades. The methanol-to-olefins (MTO) is an interesting reaction to produce those alkenes using coal, gas or alternative sources, like biomass, through syngas as a source for the production of methanol. This technology has been widely applied since 1985 and most of the processes are making use of zeolites as catalysts, particularly ZSM-5. Although its selectivity is not especially biased over light olefins, it resists to a quick deactivation by coke deposition, making it quite attractive when it comes to industrial environments; nevertheless, this is a highly exothermic reaction, which is hard to control and to anticipate problems, such as temperature runaways or hot-spots, inside the catalytic bed. The main focus of this project is to study those temperature effects, by addressing both experimental, where the catalytic performance and the temperature profiles are studied, and modelling fronts, which consists in a five step strategy to predict the weight fractions and activity. The mind-set of catalytic testing is present in all the developed assays. It was verified that the selectivity towards light olefins increases with temperature, although this also leads to a much faster catalyst deactivation. To oppose this effect, experiments were carried using a diluted bed, having been able to increase the catalyst lifetime between 32% and 47%. Additionally, experiments with three thermocouples placed inside the catalytic bed were performed, analysing the deactivation wave and the peaks of temperature throughout the bed. Regeneration was done between consecutive runs and it was concluded that this action can be a powerful means to increase the catalyst lifetime, maintaining a constant selectivity towards light olefins, by losing acid strength in a steam stabilised zeolitic structure. On the other hand, developments on the other approach lead to the construction of a raw basic model, able to predict weight fractions, that should be tuned to be a tool for deactivation and temperature profiles prediction.

Evaluation of parasitological examination, kDNA polymerase chain reaction and rK39-based immunochromatography for the diagnosis of visceral leishmaniasis in seropositive dogs from the screening-culling program in Brazil

Introduction Dogs play a primary role in the zoonotic cycle of visceral leishmaniasis (VL). Therefore, the accurate diagnosis of infected dogs, primarily asymptomatic dogs, is crucial to the efficiency of VL control programs. Methods We investigated the agreement of four diagnostic tests for canine visceral leishmaniasis (CVL): parasite detection, either after myeloculture or by direct microscopic examination of tissue imprints; kinetoplast-deoxyribonucleic acid-polymerase chain reaction (kDNA-PCR); and an immunochromatographic test (ICT). An enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence test (IFAT), both of which were adopted as part of the screening-culling program in Brazil, were used as reference tests. Our sample set consisted of 44 seropositive dogs, 25 of which were clinically asymptomatic and 19 were symptomatic for CVL according to ELISA-IFAT. Results The highest and lowest test co-positivities were observed for ICT (77.3%) and myeloculture (58.1%), respectively. When analyzed together, the overall percentage of co-positive tests was significantly higher for the symptomatic group compared to the asymptomatic group. However, only ICT was significantly different based on the results of a separate analysis per test for each group of dogs. The majority (93.8%) of animals exhibited at least one positive test result, with an average of 2.66 positive tests per dog. Half of the symptomatic dogs tested positive for all four tests administered. Conclusions The variability between test results reinforces the need for more efficient and reliable methods to accurately diagnose canine VL, particularly in asymptomatic animals.

Leishmania, Babesia and Ehrlichia in urban pet dogs: co-infection or cross-reaction in serological methods?

INTRODUCTION: The present study was designed to assess the occurrence of co-infection or cross-reaction in the serological techniques used for detecting the anti-Leishmania spp., -Babesia canis vogeli and -Ehrlichia canis antibodies in urban dogs from an area endemic to these parasites. METHODS: The serum samples from dogs were tested for the Babesia canis vogeli strain Belo Horizonte antigen and Ehrlichia canis strain São Paulo by immunofluorescence antibody test (IFAT) and by anti-Leishmania immunoglobulin G (IgG) antibody detection to assess Leishmania infection. We used the following four commercial kits for canine visceral leishmaniasis: ELISA, IFAT, Dual Path Platform (DPP) (Bio Manguinhos(r)/FIOCRUZ/MS) and a rK39 RDT (Kalazar Detect Canine Rapid Test; Inbios). RESULTS : Of 96 serum samples submitted to serological assays, 4 (4.2%) were positive for Leishmania as determined by ELISA; 12 (12.5%), by IFAT; 14 (14.6%) by rK39 RDT; and 20 (20.8%), by DPP. Antibodies against Ehrlichia and Babesia were detected in 23/96 (23.9%) and 30/96 (31.2%) samples, respectively. No significant association was identified between the results of tests for detecting Babesia or Ehrlichia and those for detecting Leishmania (p-value>0.05). CONCLUSIONS: In the present study, we demonstrated co-infection with Ehrlichia or Babesia and Leishmania in dogs from Minas Gerais (Brazil); we also found that the serological tests that were used did not cross-react.

Structural connectivity based on diffusion Kurtosis imaging

Structural connectivity models based on Diffusion Tensor Imaging (DTI) are strongly affected by the technique’s inability to resolve crossing fibres, either intra- or inter-hemispherical connections. Several models have been proposed to address this issue, including an algorithm aiming to resolve crossing fibres which is based on Diffusion Kurtosis Imaging (DKI). This technique is clinically feasible, even when multi-band acquisitions are not available, and compatible with multi-shell acquisition schemes. DKI is an extension of DTI enabling the estimation of diffusion tensor and diffusion kurtosis metrics. In this study we compare the performance of DKI and DTI in performing structural brain connectivity. Six healthy subjects were recruited, aged between 25 and 35 (three females). The MRI experiments were performed using a 3T Siemens Trio with a 32-channel head coil. The scans included a T1-weighted sequence (1mm3), and a DWI with b-values 0, 1000 and 2000 s:mm

Polymerase chain reaction-based method for the identification of Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in mucosal tissues conserved in paraffin

ABSTRACTINTRODUCTION: In the Americas, mucosal leishmaniasis is primarily associated with infection by Leishmania (Viannia) braziliensis. However, Leishmania (Viannia) guyanensis is another important cause of this disease in the Brazilian Amazon. In this study, we aimed at detecting Leishmaniadeoxyribonucleic acid (DNA) within paraffin-embedded fragments of mucosal tissues, and characterizing the infecting parasite species.METHODS: We evaluated samples collected from 114 patients treated at a reference center in the Brazilian Amazon by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses.RESULTS: Direct examination of biopsy imprints detected parasites in 10 of the 114 samples, while evaluation of hematoxylin and eosin-stained slides detected amastigotes in an additional 17 samples. Meanwhile, 31/114 samples (27.2%) were positive for Leishmania spp. kinetoplast deoxyribonucleic acid (kDNA) by PCR analysis. Of these, 17 (54.8%) yielded amplification of the mini-exon PCR target, thereby allowing for PCR-RFLP-based identification. Six of the samples were identified as L. (V.) braziliensis, while the remaining 11 were identified as L. (V.) guyanensis.CONCLUSIONS: The results of this study demonstrate the feasibility of applying molecular techniques for the diagnosis of human parasites within paraffin-embedded tissues. Moreover, our findings confirm that L. (V.) guyanensisis a relevant causative agent of mucosal leishmaniasis in the Brazilian Amazon.

Development and modulation of magnetic responsive supported ionic liquid membranes

The work presented in this thesis aims at developing a new separation process based on the application of supported magnetic ionic liquid membranes, SMILMs, using magnetic ionic liquids, MILs. MILs have attracted growing interest due to their ability to change their physicochemical characteristics when exposed to variable magnetic field conditions. The magnetic responsive behavior of MILs is thus expected to contribute for the development of more efficient separation processes, such as supported liquid membranes, where MILs may be used as a selective carrier. Driven by the MILs behavior, these membranes are expected to switch reversibly their permeability and selectivity by in situ and non-invasive adjustment of the conditions (e.g. intensity, direction vector and uniformity) of an external applied magnetic field. The development of these magnetic responsive membrane processes were anticipated by studies, performed along the first stage of this PhD work, aiming at getting a deep knowledge on the influence of magnetic field on MILs properties. The influence of the magnetic field on the molecular dynamics and structural rearrangement of MILs ionic network was assessed through a 1H-NMR technique. Through the 1H-NMR relaxometry analysis it was possible to estimate the self-diffusion profiles of two different model MILs, [Aliquat][FeCl4] and [P66614][FeCl4]. A comparative analysis was established between the behavior of magnetic and non-magnetic ionic liquids, MILs and ILs, to facilitate the perception of the magnetic field impact on MILs properties. In contrast to ILs, MILs show a specific relaxation mechanism, characterized by the magnetic dependence of their self-diffusion coefficients. MILs self-diffusion coefficients increased in the presence of magnetic field whereas ILs self-diffusion was not affected. In order to understand the reasons underlying the magnetic dependence of MILs self-diffusion, studies were performed to investigate the influence of the magnetic field on MILs’ viscosity. It was observed that the MIL´s viscosity decreases with the increase of the magnetic field, explaining the increase of MILs self-diffusion according to the modified Stokes- Einstein equation. Different gas and liquid transport studies were therefore performed aiming to determine the influence of the magnetic behavior of MILs on solute transport through SMILMs. Gas permeation studies were performed using pure CO2 andN2 gas streams and air, using a series of phosphonium cation based MILs, containing different paramagnetic anions. Transport studies were conducted in the presence and absence of magnetic field at a maximum intensity of 1.5T. The results revealed that gas permeability increased in the presence of the magnetic field, however, without affecting the membrane selectivity. The increase of gas permeability through SMILMs was related to the decrease of the MILs viscosity under magnetic field conditions.(...)

Biotransformation/separation routes to obtain pure enantiomers

The objective of the work presented in this thesis was the development of an innovative approach for the separation of enantiomers of secondary alcohols, combining the use of an ionic liquid (IL) - both as solvent for conducting enzymatic kinetic resolution and as acylating agent - with the use of carbon dioxide (CO2) as solvent for extraction. Menthol was selected for testing this reaction/separation approach due to the increasing demand for this substance, which is widely used in the pharmaceutical, cosmetics and food industries. With a view to using an ionic ester as acylating agent, whose conversion led to the release of ethanol, and due to the need to remove this alcohol so as to drive reaction equilibrium forward, a phase equilibrium study was conducted for the ehtanol/(±)-menthol/CO2 system, at pressures between 8 and 10 MPa and temperatures between 40 and 50 oC. It was found that CO2 is more selective towards ethanol, especially at the lowest pressure and highest temperature tested, leading to separation factors in the range 1.6-7.6. The pressure-temperature-composition data obtained were correlated with the Peng-Robinson equation of state and the Mathias-Klotz-Prausnitz mixing rule. The model fit the experimental results well, with an average absolute deviation (AAD) of 3.7 %. The resolution of racemic menthol was studied using two lipases, namely lipase from Candida rugosa (CRL) and immobilized lipase B from Candida antarctica (CALB), and two ionic acylating esters. No reaction was detected in either case. (R,S)-1-phenylethanol was used next, and it was found that with CRL low, nonselective, conversion of the alcohol took place, whereas CALB led to an enantiomeric excess (ee) of the substrate of 95%, at 30% conversion. Other acylating agents were tested for the resolution of (±)-menthol, namely vinyl esters and acid anhydrides, using several lipases and varying other parameters that affect conversion and enantioselectivity, such as substrate concentration, solvent and temperature. One such acylating agent was propionic anhydride. It was thus performed a phase equilibrium study on the propionic anhydride/CO2 system, at temperatures between 35 and 50 oC. This study revealed that, at 35 oC and pressures from 7 MPa, the system is monophasic for all compositions. The enzymatic catalysis studies carried out with propionic anhydride revealed that the extent of noncatalyzed reaction was high, with a negative effect on enantioselectivity. These studies showed also that it was possible to reduce considerably the impact of the noncatalyzed reaction relative to the reaction catalyzed by CRL by lowering temperature to 4 oC. Vinyl decanoate was shown to lead to the best results at conditions amenable to a process combining the use of supercritical CO2 as agent for post-reaction separation. The use of vinyl decanoate in a number of IL solvents, namely [bmim][PF6], [bmim][BF4], [hmim][PF6], [omim][PF6], and [bmim][Tf2N], led to an enantiomeric excess of product (eep) values of over 96%, at about 50% conversion, using CRL. In n-hexane and supercritical CO2, reaction progressed more slowly.(...)

Simulation of humidity fields in concrete: experimental validation and parameter estimation

The moisture content in concrete structures has an important influence in their behavior and performance. Several vali-dated numerical approaches adopt the governing equation for relative humidity fields proposed in Model Code 1990/2010. Nevertheless there is no integrative study which addresses the choice of parameters for the simulation of the humidity diffusion phenomenon, particularly in concern to the range of parameters forwarded by Model Code 1990/2010. A software based on a Finite Difference Method Algorithm (1D and axisymmetric cases) is used to perform sensitivity analyses on the main parameters in a normal strength concrete. Then, based on the conclusions of the sensi-tivity analyses, experimental results from nine different concrete compositions are analyzed. The software is used to identify the main material parameters that better fit the experimental data. In general, the model was able to satisfactory fit the experimental results and new correlations were proposed, particularly focusing on the boundary transfer coeffi-cient.

Grafting of adipic anhydride to carbon nanotubes through a Diels-Alder cycloaddition/oxidation cascade reaction

Accepted Manuscript

Modelação, simulação e implementação de agitação acústica para aplicações em microfluídica

Tese de Doutoramento (Programa Doutoral em Engenharia Biomédica)